Promoters for sugarcane transformation : isolation of specific sequences and evaluation of rolC.

Abstract

Increasing the sucrose yield and the disease resistance of plants are two major objectives of the transgenic sugarcane plant programme in South Africa. The sugarcane culm has thus been identified as one of the main target areas for transgene expression. A shortage of reliable promoter elements as well as patent limitations have necessitated the isolation of promoters that are preferentially expressed in the sugarcane culm. In the present study two different approaches were followed to isolate such promoters, and the bacterial promoter, rolC, was evaluated for tissue-specific expression in sugarcane. Differential display is a non-directed technique that was used to identify genes that are differentially expressed in the mature sugarcane culm. The original method was modified, and four putative culm-preferential fragments were isolated. Sequence and hybridisation analyses revealed that these fragments were false positives, and could therefore not be used to obtain a culm-specific promoter. Activity of the Agrobacterium rolC promoter was evaluated by analysing expression patterns of two reporter genes in the mature culm of transgenic sugarcane plants. Nucleic acid analyses indicated that the foreign DNA was incorporated into the sugarcane genome, and that mRNA transcripts were produced. Histochemical analysis was done to visualise rolC-driven GUS and GFP expression in the mature sugarcane culm. In both cases the reporter gene expression was restricted to the vascular bundles and specifically to the phloem. A directed approach was followed to isolate the gene and subsequently the promoter of the β-subunit of pyrophosphate-dependent phosphofructokinase (PFP-β). An incomplete cDNA clone was obtained from a mature culm cDNA library, and was used for the screening of a sugarcane genomic library. Two clones containing different parts of the PFP-β gene were isolated. A Deletion Factory™ system was used to analyse the clone containing the 5' end of the gene. The first five exons and 1747 bp of the 5' flanking region of the gene were sequenced. Preliminary activity analysis of the promoter region was done by constructing two expression vectors, and analysing transient GUS expression in sugarcane callus. Results indicated that the promoter is capable of driving foreign gene expression in callus. Transient expression levels were lower than that of the maize Ubi-1 promoter. Further analysis of the 5' flanking region will be done to establish whether cis-acting elements outside the analysed area have an influence on the activity of the promoter.